The effects of oxygen concentration and reoxygenation fol ¥owing hypoxic cultur巴ontransforming growth factor-s I (TGF-s1)巴xpr巴ssionand types 1 and II collagen [α2(1) and α1 (II)] mRNA expression were巴xaminedin neonatal rat cardiac fibroblasts cu¥tur巴dwith medium containing 10% or 1 % fetal calf serum at confluency. The oxygen tension was varied using a N2-C02-02 controllable incubator. Cells were exposed to continuous hypoxia (2% O2) or were reoxygenated (21 % O2) after hypoxic culture. Th巴TGF-s1 protein巴xpressionlevel was determined by an enzyme-linked immunosorbent assay, and the expression levels of TGF-s 1,α2(1) and α1 (II) mRNA were measur巴dby a Northern blot analysis. Both the TGF-sI mRNA and protein levels were higher in the continuous hypoxic cultures than in the continuous normoxic cultures. Their levels in reoxygenation cultures were not substantial¥y different from those in th巴continuousnormoxic cu1tures. Similarly, the expressions of both d2(1) and α1 (II) mRNA wer巴巴nhancedin continuous hypoxic culture, and no differences wer巴obtainedbetween normoxic and reoxygenation cultures. The present results indicate that hypoxia induces high expression levels of TGF-s 1 and types 1 and II collagen in rat cardiac fibroblasts and that reoxygenation does not further enhance this hypoxia-induced upregulation.

Heart rate (HR) variability is an important factor for the prognosis of heart disease. We examined the effects of cilostazol, a quinolone derivative, on HR and HR variability in patients with sick sinus syndrome.

Mucosa‐associated lymphoid tissue (MALT) lymphomata observed simultaneously in the stomach and colon are rare. We report concurrent gastric and colonic low‐grade MALT lymphomata that originated from the same clone in a 58‐year‐old Japanese man without Helicobacter pylori infection. Endoscopy showed multiple erosive lesions in the gastric body and antrum, and a single flat elevation with an irregular margin in the sigmoid colon. Histopathological findings of both lesions suggested low‐grade MALT lymphoma. Lymphoepithelial lesions were evident in the gastric lesions, but not in the colonic lesion. Southern blot analysis of lymphoma cells revealed the same immunoglobulin heavy‐chain rearrangement pattern. The chromosomal translocation t(11;18)(q21;q21) was also observed. After six courses of cyclophosphamide, doxorubicin, vincristine and predonisolone, the gastric lesions disappeared endoscopically, while the colonic lesion persisted. A sigmoidectomy was consequently performed. The chromosomal translocation may be related to the pathogenesis of the present MALT lymphoma case without H. pylori infection. It is interesting that the gastric and colonic lesions differed in response to treatment and in their endoscopic and histologic features, despite having the same origin.

Although primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) are two independent autoimmune liver diseases, it is sometimes difficult to characterize the variant forms of autoimmune liver disease. A PBC scoring system, in combination with the AIH scoring system may be helpful to characterize such patients. Methods: A PBC scoring system was introduced that selected 14 categories characteristic of PBC. One hundred and thirty-four patients with PBC, 31 patients with autoimmune cholangitis (AIC), 22 patients with overlap syndrome, and 48 patients with AIH were included in the study. The AIC patients fulfilled the PBC criteria but were negative for anti-mitochondrial antibody and positive for anti-nuclear antibody. Overlap syndrome patients fulfilled both the PBC and AIH criteria. Results: The total scores (means ± SD) for the PBC, AIC, overlap syndrome, and AIH patients were 23.3 ± 4.7, 9.3 ± 4.4, 18.0 ± 5.9, and 3.6 ± 3.3, respectively. When definite and probable PBC patients were defined as those with a total score of over 17 and 9–17, respectively, all except for 1 patient could be classified as definite or probable PBC. Four of the 48 AIH patients were classified as probable PBC. PBC scores for the variant autoimmune liver diseases showed a wide deviation. Plotting both PBC and AIH scores in a rectangular coordinate enabled us to locate each patient with variant forms according to the deviation from classical PBC or AIH. Conclusions: The PBC scoring system might be useful in characterizing the features of variant forms of autoimmune liver disease.

Transforming growth factor-beta1 (TGF-beta1) exerts potent immunosuppressive effects. In this study, we investigated the potential role of TGF-beta1 produced by hepatocellular carcinoma (HCC) cell lines in immunosuppression mechanisms. Using the Mv1Lu cell-growth inhibition assay and an enzyme-linked immunosorbent assay (ELISA), we detected optimal levels of TGF-beta1 in the culture supernatants conditioned by the HCC cell lines PLC/PRF/5, Hep3B, and HepG2. To determine the biological activity of TGF-beta1 in the supernatants, we examined the effects of the culture supernatants on the production of interferon (IFN)-gamma induced during the culture of peripheral blood mononuclear cells (PBMCs) stimulated with interleukin (IL)-12. IFN-gamma production of IL-12-stimulated PBMCs in the 1:1 dilution of the acid-activated conditioned medium of PLC/PRF/5, Hep3B, and HepG2 reduced to 14.7 +/- 0.8, 17.3 +/- 9.0, and 35.9 +/- 14.6%, respectively, compared with the value in the culture with control medium (complete culture medium). These results suggest that HCC cells producing TGF-beta1 may reduce the generation or activation of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, and thus could enhance their ability to escape immune-mediated surveillance.

症例は44歳,女性.肝機能異常の精査,治療目的に当科に紹介入院となった.入院時,高度肝機能異常を示し,各種肝炎ウイルスマーカー陰性,抗核抗体陽性,血清IgG値高値であり,自己免疫性肝炎と診断した.プロトロンビン時間(活性)は30%と低下し,症状発現後18日目に肝性昏睡III度の脳症が出現した.血漿交換等の内科的治療では肝機能は改善せず,救命困難と考え,第54病日に夫をドナーとして生体部分肝移植を施行した.本症例は自己免疫性肝炎の劇症化で,しかも生体部分肝移植により救命しえた症例であり,今回報告する.

. Objectives: Dermatopontin, a 22 kDa extracellular matrix (ECM) protein, has been shown to interact with other ECM components, especially decorin, and to regulate ECM formation. We examined dermatopontin mRNA expression in the myocardial infarct zone. Methods: The cDNA encoding the rat dermatopontin was cloned by RT-PCR based on screening results from the Expressed Sequence Tag™ database. The dermatopontin mRNA expression was examined in the infarct zone after experimentally induced myocardial infarction in rats by the methods of Northern blotting and in situ hybridization. The expression of dermatopontin mRNA was compared to that of decorin and type I collagen mRNAs. Results: The isolated clone contained a 609 bp cDNA insert containing a complete open reading frame encoding 202 amino acids. The rat dermatopontin cDNA showed high homology to human and mouse counterparts (>96 %). Northern blotting demonstrated that dermatopontin mRNA expression did not markedly increase on day 2, but was increased on days 7, 14 and 28 by 2.4-, 4.1- and 4.2-fold, respectively, compared to that in preligation hearts. Dermatopontin mRNA expression was regulated almost in parallel with decorin mRNA expression. In situ hybridization demonstrated mRNA signals for dermatopontin in macrophages and spindle-shaped mesenchymal cells (fibroblasts and myofibroblasts) located in the infarct interior zone around infarcted necrotic tissue on day 7. Coexpression of dermatopontin mRNA with decorin and type I collagen mRNAs was observed in spindle-shaped mesenchymal cells. Conclusions: The present results demonstrated the time-dependent increase in the expression of dermatopontin mRNA in parallel with that of decorin mRNA in the infarct zone. Coexpression of dermatopontin mRNA with decorin and type I collagen mRNAs suggests that dermatopontin plays a role in ECM (fibrillar collagen matrix) reformation in the infarct along with decorin and type I collagen.

Ventricular remodeling is a major determinant of the long-term prognosis of patients with acute myocardial infarction (AMI). No previous study examined the relation of ST-segment re-elevation to left ventricular (LV) volume and function in patients with successful reperfusion. We examined the relation of ST-segment re-elevation to LV function and volume indices in 51 patients with anterior wall AMI who underwent successful reperfusion by direct coronary angioplasty. A 12-lead electrocardiogram was recorded once a day until 7 days after the onset of AMI. ST-segment shift was measured and SigmaST was defined as the sum of ST-segment elevation obtained from leads V2, V3, and V4. ST-segment re-elevation was defined as present when the difference between maximal and minimal SigmaST (DeltaST) was >0.3mV. LV indices were obtained from left ventriculography performed approximately 1 month after the onset of AMI. ST-segment re-elevation was observed in 15 patients (29%). No significant differences were observed between the ST- re-elevation group and non-ST-re-elevation group in LV ejection fraction (49.4 +/- 14.0 vs. 51.2 +/- 11.5%), LV end-systolic volume index (35.8 +/- 13.1 vs. 33.8 +/- 12.5 mL/m(2)) or LV end-diastolic volume index (69.7 +/- 12.8 vs. 68.3 +/- 14.4 mL/m(2)). The difference between maximal and minimal SigmaST (DeltaST) was not significantly correlated with any LV index examined. In conclusion, the present study revealed that ST-segment re-elevation after successful reperfusion in anterior wall AMI patients was not related to LV volume or function, indicating that ST-re-elevation is not a clinically meaningful indicator of LV remodeling.

Heart rate (HR) variability is important with respect to disease prognosis and the effects of drugs. Lorenz scatterplots provide a simple way to evaluate HR variability visually. The relation of Lorenz scatterplots to frequency-domain HR variability was examined in 75 Holter recordings and in simulated HR trends. The length of Lorenz scatterplots was double-exponentially correlated with total frequency and very-low frequency powers, with correlation coefficients (r) of 0.88. The width of Lorenz scatterplots was highly correlated with the high frequency power (r = 0.98). The sum of the width and length of Lorenz scatterplots was highly correlated with the total frequency power (r = 0.92). Identical results were obtained when simulated HR trends were used. In conclusion, Lorenz scatterplots provide a simple way to estimate the frequency-domain HR variability.

In the present study, we have analyzed the amount of precore wild-type hepatitis B virus (HBV) (wild-type) and precore mutant HBV (nt 1896: G-->A) (precore mutant) of HBV carriers; 31 asymptomatic healthy carriers (ASC) and 28 patients with chronic hepatitis (CH). Wild-type and precore mutant were quantified using sensitive and specific quantification methods: competitive wild-type-sequence-specific assay and competitive mutation-site-specific assay with different sets of specific primers and internal controls. Median serum levels of wild-type and precore mutant were 9.60 and 8.60 log copies/ml (median percentages of precore mutant in total HBV-DNA: 11.7%) in HBeAg(+) ASC, 8.48 and 8.00 (33.3%) in HBeAg(+) CH, and 6.30 and 6.85 (84.7%) in anti-HBe(+) CH, respectively, showing higher levels of the relative amount of precore mutant to wild-type along with HBeAg/anti-HBe status. Only precore mutant, but not wild-type was detected in anti-HBe(+) ASC. Although median percentages of precore mutant at the anti-HBe(+) ASC and CH stages were much higher than those at the HBeAg(+) ASC and CH stages, a substantial amount of precore mutant was found even at the HBeAg(+) stages. Existence of a substantial amount of precore mutant even in HBeAg(+) ASC suggests that the occurrence of precore mutant is not always closely associated with seroconversion from HBeAg to anti-HBe.

In this study, we assessed whether standard doses of antiulcer drugs rebamipide, and ecabet sodium influence the accuracy of the urea breath test (UBT). Without medication, values of UBT estimated with nondispersive isotope-selective infrared spectrometry did not significantly change during follow-up (average 4.4 months). Then, we randomized 21 H. pylori-positive patients without serious disease to receive either rebamipide or ecabet sodium for 28 days. UBT was performed at baseline, on day 14, on day 28, and 2 weeks after cessation of drugs. On day 14, the median value of UBT declined significantly (p = 0.0113) compared to baseline with ecabet sodium but not with rebamipide. This caused a false-negative result of UBT in one of 11 patients who received ecabet sodium. The decline of UBT resolved 2 weeks after drug cessation. In addition, we performed a crossover study in 11 H. pylori-positive volunteers with these drugs, and ecabet sodium but not rebamipide significantly reduced UBT values (p = 0.0058). These findings indicate that ecabet sodium adversely influences the accuracy of UBT and that the withdrawal of this drug before testing appears to be necessary to avoid false-negative results.

Patients with PBC produce a directed, specific response to a single immunodominant autoepitope of PDC-E2 within the inner lipoyl domain. In contrast, immunized animals react to multiple epitopes and rarely recognize the inner lipoyl domain. In other autoimmune diseases, apoptosis plays a critical role in antigen presentation; the caspases and granzyme B are the key proteases in the generation of autoepitopes. To determine the specific cleavage pattern of full-length recombinant PDC-E2, we performed in vitro digestion with caspases-3, -6, -8 and granzyme B. The resulting fragments were immunoblotted and probed with an extensive panel of monoclonal anti-PDC-E2 antibodies and sera from patients with PBC. Interestingly, on granzyme B digestion, PDC-E2 lost reactivity, suggesting the destruction of the immunodominant epitope. Because this site contains the major epitope for both B cells and T cells, it suggests that granzyme B is unlikely to be involved in generation of autoepitopes in primary biliar cirrhosis (PBC). In contrast, following treatment with the caspase enzymes, immunoreactive fragments were generated. Indeed, by confocal microscopy, activated caspase-3 is found in the marginal hepatocytes and bile ducts. Moreover, caspase-3 staining was strongest in the small intrahepatic bile ducts, the major site of tissue destruction in PBC. In conclusion, these data suggest that following apoptosis, the caspase family of proteolytic enzymes have the potential to generate immunogenic fragments that contribute to the autoantigen reservoir and the production of antimitochondrial antibodies. These findings are also consistent with the generation of an autoimmune response against an intracellular antigen that evades catabolism during apoptosis.

In fulminant hepatic failure (FHF), rapidly progressive cerebral oedema remains the main fatal complication and an obstacle in liver transplantation. A 29-year-old Japanese woman presented with sudden-onset hepatic encephalopathy and jaundice. Hepatic encephalopathy deteriorated within 2 days of the onset of jaundice. She manifested extensory sustained clonus and was responsive only to pain. Diffuse cerebral oedema was noted on brain computerized tomography (CT) scan. Urgent living-donor liver transplantation (LDLT) at the time of admission was abandoned because of deterioration of neurological status and radiologically evident diffuse cerebral oedema. Instead, a high-flow-rate (7.2-9.0 l/h) haemodiafiltration with a high-performance membrane was commenced, combined with plasma exchange. This treatment regimen resulted in a gradual improvement of hepatic encephalopathy and complete disappearance of cerebral oedema within 7 days. Liver regeneration did not occur during this period, as evident by CT scan volumetry and serological tests. LDLT was subsequently performed using the right liver lobe of the patient's brother. Our case suggests that high-flow-rate haemodiafiltration with a high-performance membrane, combined with plasma exchange, could potentially be brain-support therapy for patients with FHF, and may contribute, when combined with liver transplantation, to the improvement of prognosis in hyperacute FHF.

To elucidate the positive frequency of hepatitis B virus (HBV) DNA in patients with hepatitis B surface antigen (HBsAg) and antibody to hepatitis C virus (HCVAb) negative (NBNC) chronic liver disease, we detected serum HBV-DNA using the real-time detection polymerase chain reaction method (RTD-PCR) in comparison with HBsAg and HCVAb negative individuals with normal alanine aminotransferase (ALT). We also revealed the nucleotide sequence of the X-pre-C gene-coding region of intrahepatic HBV-DNA. Serum HBV-DNA was detected in 12 of 36 NBNC patients (five of 22 chronic hepatitis, three of eight liver cirrhosis and four of six hepatocellular carcinoma), whereas none of 31 control individuals were positive for serum HBV-DNA (P<0.01). Serum antibody to hepatitis B core antigen (HBcAb) was positive in nine of 36 in patients with NBNC patients and was positive in nine of 31 in control individuals (n.s). Five of six NBNC patients with serum HBV-DNA positive by RTD-PCR were also positive for intrahepatic HBV-DNA. Analysis of the nucleotide sequence of the X-pre-C coding region of HBV-DNA in the liver showed similar mutation sites of 1677, 1679, 1709, 1753, 1762, 1764. These findings suggested that serum HBV-DNA was more frequently detected in NBNC patients than normal individuals and the presence of low levels of serum HBV-DNA was correlated to the 1677–1764 X-pre-C mutations.

We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE). Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B), anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1), a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin), a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside), and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA), Ricinus communis Agglutinin I (RCA-I), and Concanavalin A (ConA) showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is mitogenic and chemotactic for many cell types. HB-EGF is induced in pathological states which require cell mitogenesis and proliferation, including angiogenesis, and has been reported to interact functionally with basic fibroblast growth factor (bFGF). To test our hypothesis that HB-EGF mRNA expression is increased in myocardial infarction, we used Northern hybridization in rats to investigate the expression of HB-EGF and EGF receptor mRNAs expression in the infarct zone compared to the expression of bFGF and FGF receptor mRNAs. We also performed in situ hybridization to identify the cells responsible for HB-EGF mRNA production. HB-EGF mRNA rapidly increased after ligation (mean ± SE, 5.6 ± 0.23-fold increase at 6 hours compared to the preligation heart levels) and reached a maximum level (9.1 ± 0.42-fold increase) around 12 hours. HB-EGF mRNA then gradually decreased on day 1 (5.8 ± 1.0-fold increase), day 2 (3.2 ± 0.94-fold increase) and day 3 (1.9 ± 0.33-fold increase) after ligation. Parallel changes in bFGF mRNA expression were observed (6, 12 hours, days 1, 2 and 3; 3.6 ± 0.42-, 5.3 ± 0.12-, 2.3 ± 0.12-, 1.7 ± 0.03- and 0.95 ± 0.03-fold increase, respectively). EGF receptor (ErbB-1) mRNA was gradually increased on day 2 (2.4 ± 0.53-fold increase), day 7(4.0 ± 0.61-fold increase) and day 14 (7.0 ± 0.61-fold increase). Similarly, FGF receptor (FGF receptor-1) mRNA was gradually increased (days 2, 7 and 14; 1.3 ± 0.13-, 1.5 ± 0.17- and 2.3 ± 0.15-fold increase, respectively). Reperfusion after a 2-hour ligation (too late to salvage myocytes) enhanced HB-EGF (12 hours, 16.8 ± 1.8-fold increase) and bFGF (12 hours, 10.4 ± 1.1-fold increase) mRNA expression. The cells responsible for the increased production of HB-EGF mRNA were shown by in situ hybridization to be surviving myocytes located in the infarct peripheral zone around infarct necrotizing tissue. In conclusion, our results demonstrated a rapid increase in HB-EGF mRNA expression concomitant with an increase in bFGF mRNA expression, suggesting that HB-EGF and bFGF might play some role in the course of pathological changes in the infarct in the early inflammatory phase. Reperfusion at times too late to salvage myocytes accelerated sequential changes in the expression of both HB-EGF and bFGF mRNAs.